Small-molecule AMP-activated Protein Kinase Activator Research Articles

AMP-activated protein kinase activation suppresses leptin expression independently of adipogenesis in primary murine adipocytes.

Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

ObjectiveThe metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. MethodsGlobal AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. ResultsGenetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. ConclusionsThese results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

AMPK activation by A-769662 and 991 does not affect catecholamine-induced lipolysis in human adipocytes.

Activation of AMP-activated protein kinase (AMPK) is considered an attractive strategy for the treatment of type 2 diabetes. Favorable metabolic effects of AMPK activation are mainly observed in skeletal muscle and liver tissue, whereas the effects in human adipose tissue are only poorly understood. Previous studies, which largely employed the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), suggest an antilipolytic role of AMPK in adipocytes. The aim of this work was to reinvestigate the role of AMPK in the regulation of lipolysis, using the novel allosteric small-molecule AMPK activators A-769662 and 991, with a focus on human adipocytes. For this purpose, human primary subcutaneous adipocytes were treated with A-769662, 991, or AICAR, as a control, before being stimulated with isoproterenol. AMPK activity status, glycerol release, and the phosphorylation of hormone-sensitive lipase (HSL), a key regulator of lipolysis, were then monitored. Our results show that both A-769662 and 991 activated AMPK to a level that was similar to, or greater than, that induced by AICAR. In contrast to AICAR, which as expected was antilipolytic, neither A-769662 nor 991 affected lipolysis in human adipocytes, although 991 treatment led to altered HSL phosphorylation. Furthermore, we suggest that HSL Ser660 is an important regulator of lipolytic activity in human adipocytes. These data suggest that the antilipolytic effect observed with AICAR in previous studies is, at least to some extent, AMPK independent.

AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development

Nonalcoholic fatty liver disease is a highly prevalent component of disorders associated with disrupted energy homeostasis. Although dysregulation of the energy sensor AMP-activated protein kinase (AMPK) is viewed as a pathogenic factor in the development of fatty liver its role has not been directly demonstrated. Unexpectedly, we show here that liver-specific AMPK KO mice display normal hepatic lipid homeostasis and are not prone to fatty liver development, indicating that the decreases in AMPK activity associated with hepatic steatosis may be a consequence, rather than a cause, of changes in hepatic metabolism. In contrast, we found that pharmacological re-activation of downregulated AMPK in fatty liver is sufficient to normalize hepatic lipid content. Mechanistically, AMPK activation reduces hepatic triglyceride content both by inhibiting lipid synthesis and by stimulating fatty acid oxidation in an LKB1-dependent manner, through a transcription-independent mechanism. Furthermore, the effect of the antidiabetic drug metformin on lipogenesis inhibition and fatty acid oxidation stimulation was enhanced by combination treatment with small-molecule AMPK activators in primary hepatocytes from mice and humans. Overall, these results demonstrate that AMPK downregulation is not a triggering factor in fatty liver development but in contrast, establish the therapeutic impact of pharmacological AMPK re-activation in the treatment of fatty liver disease.

Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase found in nearly all eukaryotes that functions as a master energy sensor in cells. During times of cell stress and changes in the AMP/ATP ratio, AMPK becomes activated and phosphorylates a multitude of protein substrates involved in various cellular processes such as metabolism, cell growth and autophagy. The endogenous ligand AMP is known to bind to the γ-subunit and activates the enzyme via three distinct mechanisms (1) enhancing phosphorylation by upstream kinases of Thr172 in the activation loop (a site critical for AMPK activity), (2) protecting Thr172 from dephosphorylation by phosphatases, and (3) allosteric activation of the kinase activity. Given the important regulatory role for AMPK in various cellular processes and the multiple known modes of activation, there is great interest in identifying small-molecule activators of this kinase and a need for assays to identify and characterize compounds. Here we describe several assay formats that have been used for identifying and characterizing small-molecule AMPK activators.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: Identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets.

A-769662 potentiates the effect of other AMP-activated protein kinase activators on cardiac glucose uptake.

AMP-activated protein kinase (AMPK), a key cellular sensor of energy, regulates metabolic homeostasis and plays a protective role in the ischemic or diabetic heart. Stimulation of cardiac glucose uptake contributes to this AMPK-mediated protection. The small-molecule AMPK activator A-769662, which binds and directly activates AMPK, has recently been characterized. A-769662-dependent AMPK activation protects the heart against an ischemia-reperfusion episode but is unable to stimulate skeletal muscle glucose uptake. Here, we tried to reconcile these conflicting findings by investigating the impact of A-769662 on cardiac AMPK signaling and glucose uptake. We showed that A-769662 promoted AMPK activation, resulting in the phosphorylation of several downstream targets, but was incapable of stimulating glucose uptake in cultured cardiomyocytes and the perfused heart. The lack of glucose uptake stimulation can be explained by A-769662's narrow specificity, since it selectively activates cardiac AMPK heterotrimeric complexes containing α2/β1-subunits, the others being presumably required for this metabolic outcome. However, when combined with classical AMPK activators, such as metformin, phenformin, oligomycin, or hypoxia, which impact AMPK heterotrimers more broadly via elevation of cellular AMP levels, A-769662 induced more profound AMPK phosphorylation and subsequent glucose uptake stimulation. The synergistic effect of A-769662 under such ischemia-mimetic conditions protected cardiomyocytes against ROS production and cell death. In conclusion, despite the fact that A-769662 activates AMPK, it alone does not significantly stimulate glucose uptake. However, strikingly, its ability of potentiating the action on other AMPK activators makes it a potentially useful participant in the protective role of AMPK in the heart.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.

Novel Small-Molecule AMP-Activated Protein Kinase Allosteric Activator with Beneficial Effects in db/db Mice

AMP-activated protein kinase (AMPK) is an energy sensor of metabolism that is an attractive therapeutic target for type 2 diabetes mellitus and metabolic syndrome. Using a homogeneous scintillation proximity assay (SPA), we identified a new small-molecule AMPK activator, ZLN024, which allosterically stimulated active AMPK heterotrimers and the inactive α1 subunit truncations α1 (1–394) and α1 (1–335) but not α1 (1–312). AMPK activation by ZLN024 requires the pre-phosphorylation of Thr-172 by at least one upstream kinase and protects AMPK Thr-172 against dephosphorylation by PP2Cα. ZLN024 activated AMPK in L6 myotubes and stimulated glucose uptake and fatty acid oxidation without increasing the ADP/ATP ratio. ZLN024 also activated AMPK in primary hepatocytes, decreased fatty acid synthesis and glucose output. Treatment of db/db mice with 15 mg/kg/day ZLN024 improved glucose tolerance; liver tissue weight, triacylglycerol and the total cholesterol content were decreased. The hepatic transcriptional level of G6Pase, FAS and mtGPAT were reduced. The transcription of genes involved in fatty acid oxidation and the mitochondrial biogenesis of muscle tissue were elevated. The ACC phosphorylation was increased in muscle and liver. This study provides a novel allosteric AMPK activator for functional study in vitro and in vivo and demonstrates that AMPK allosteric activators could be a promising therapeutic approach for type 2 diabetes mellitus and metabolic syndrome.

AMPK activation by a novel small molecule of mitochondrial regulation enhances fatty acid oxidation, ketogenesis and branched chain amino acid catabolism in vivo

The 5′‐AMP‐activated protein kinase (AMPK) is a key regulator of energy homeostasis. Pharmacologic activation of AMPK is beneficial in treating metabolic disorders and improving exercise endurance. We previously identified small molecule AMPK activators that improved glucose tolerance and running endurance. These compounds reduced cellular oxygen consumption and increased AMP/ATP and ADP/ATP ratios, demonstrating that AMPK activation is due to a change in energy status which results from altered mitochondrial respiration. Further biochemical studies showed that the target appeared to be mitochondrial complex I. In vivo metabolite profiling indicated that there was an up‐regulation of fatty acid oxidation, ketogenesis and catabolism of branched chain amino acids in the compound‐treated mice. The up‐regulation in fatty acid catabolism was supported by a clear reduction of fat pads in treated mice. Furthermore, UCP1 protein expression was increased in the white adipose tissues. Taken together, our results demonstrate beneficial outcomes by AMPK activation through mitochondrial regulation. These studies should help to provide a molecular understanding for the physiological effects of AMPK activation and for the therapeutic applications of AMPK activators.

A small molecule AMPK activator protects the heart against ischemia–reperfusion injury

AMP-activated protein kinase (AMPK) is a stress signaling enzyme that orchestrates the regulation of energy-generating and -consuming pathways. Intrinsic AMPK activation protects the heart against ischemic injury and apoptosis, but whether pharmacologic AMPK stimulation mitigates ischemia–reperfusion damage is unknown. The aims of this study were to determine whether direct stimulation of AMPK using a small molecule activator, A-769662, attenuates myocardial ischemia–reperfusion injury and to examine its cardioprotective mechanisms. Isolated mouse hearts pre-treated with A-769662 had better recovery of left ventricular contractile function (55% vs. 29% of baseline rate-pressure product; p = 0.03) and less myocardial necrosis (56% reduction in infarct size; p < 0.01) during post-ischemic reperfusion compared to control hearts. Pre-treatment with A-769662 in vivo attenuated infarct size in C57Bl/6 mice undergoing left coronary artery occlusion and reperfusion compared to vehicle (36% vs. 18%, p = 0.025). Mouse hearts with genetically inactivated AMPK were not protected by A-769662, indicating the specificity of this compound. Pre-treatment with A-769662 increased the phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2), preserved energy charge during ischemia, delayed the development of ischemic contracture, and reduced myocardial apoptosis and necrosis. A-769662 also augmented endothelial nitric oxide synthase (eNOS) activation during ischemia, which partially attenuated myocardial stunning, but did not prevent necrosis. AMPK is a therapeutic target that can be stimulated by a direct-acting small molecule in order to prevent injury during ischemia–reperfusion. The use of AMPK activators may represent a novel strategy to protect the heart and other solid organs against ischemia.

Small Molecule Antagonizes Autoinhibition and Activates AMP-activated Protein Kinase in Cells

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.

Discovery and SAR development of thienopyridones: A class of small molecule AMPK activators

AMP-activated protein kinase (AMPK) is well established as a sensor and regulator of intracellular and whole-body energy metabolism. A high-throughput screen was performed in order to identify chemotypes that are bound by AMPK. A novel thienopyridone compound ( 1) was identified and subsequently optimized. The structure–activity relationships that emerged from this effort are described.

Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome

AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC50 = 0.8 microM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC50 = 3.2 microM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.

Homogeneous assays for adenosine 5 ′-monophosphate-activated protein kinase

Allosteric activation of 5 ′-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS 133-CREB and an engineered peptide ACC-CREBp. Anti-pS 133-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl–CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS 133-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody–antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use.